Small molecule polypeptide for improving immunotherapy sensitivity and application thereof in preparation of immunotherapy adjuvant
By promoting the proliferation of CD4+ T cells and NK cells and inhibiting Treg cells through the small molecule peptide T-P28, the problem of tumor immune escape is solved, and the sensitivity and efficacy of immunotherapy are improved. In particular, the therapeutic effect on melanoma, colon cancer and esophageal cancer is significant when used in combination with PD1 inhibitors.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- SHANDONG UNIV
- Filing Date
- 2024-12-06
- Publication Date
- 2026-06-09
Smart Images
Figure CN122167536A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to a small molecule polypeptide that improves the sensitivity to immunotherapy and its application in the preparation of adjuvant drugs for immunotherapy, belonging to the field of biomedical technology. Background Technology
[0002] With the advent of precision medicine, especially the clinical application of treatments such as immunotherapy, the survival time of patients with malignant tumors has been significantly extended, and tumor cure or chronic management has become a reality. However, during immunotherapy, tumors can exhibit immune escape, and the sensitivity to immunotherapy varies considerably among different patients. Therefore, improving the sensitivity and duration of immunotherapy is an effective means to improve the survival time of cancer patients.
[0003] The efficacy of immunotherapy is closely related to the number and activation of immune cells in the tumor-associated microenvironment. T cells are the core cell type in the anti-tumor immune response, with CD4+ T cells and CD8+ T cells playing important roles. However, CD8+ T cells alone may lead to tumor immune escape. Studies have shown that B cells can influence the tumor immune response by secreting antibodies and anti-tumor cytokines. Innate immune cells also play an important role, especially M2 type tumor-associated macrophages and NK cells. However, research on adjuvant sensitizing agents for immunotherapy is currently limited.
[0004] Therefore, there is an urgent need to develop a novel adjuvant immunotherapy drug that can improve the tumor immune microenvironment, including the number and activation level of T cells, NK cells and related cytokines, chemokines, etc., and avoid similar side effects. Summary of the Invention
[0005] To address the shortcomings of existing technologies, this invention provides a small molecule polypeptide that improves immunotherapy sensitivity and its application in the preparation of adjuvant immunotherapy drugs.
[0006] The technical solution of the present invention is as follows:
[0007] A small molecule polypeptide, the amino acid sequence of which is shown in SEQ ID NO.1.
[0008] According to a preferred embodiment of the present invention, the small molecule polypeptide can be synthesized by solid-phase synthesis according to the amino acid sequence information.
[0009] The above-mentioned small molecule peptides are used in vivo and / or in vitro to promote the proliferation of CD4+ T cells and NK cells and inhibit the proliferation of Treg cells.
[0010] The above-mentioned small molecule peptides are used in the preparation of drugs to prevent and / or treat diseases by regulating CD4+ T cells, NK cells and Treg cells.
[0011] The application of the above-mentioned small molecule peptides in the preparation of adjuvant drugs for tumor immunotherapy.
[0012] According to a preferred embodiment of the present invention, the tumor is melanoma, colon cancer, or esophageal cancer.
[0013] According to a preferred embodiment of the present invention, the adjuvant drug for tumor immunotherapy is used in combination with drugs or therapies for the prevention and / or treatment of tumor immune tolerance.
[0014] According to a preferred embodiment of the present invention, the adjuvant drug for tumor immunotherapy further includes pharmaceutically acceptable carriers, excipients, additives, supplements, dispersants, or stabilizers.
[0015] According to a preferred embodiment of the present invention, the adjuvant drug for tumor immunotherapy is a solution, emulsion, suspension, or injection.
[0016] Beneficial effects:
[0017] 1. This invention is the first to discover a small molecule polypeptide that improves the sensitivity to immunotherapy. This small molecule polypeptide can promote the proliferation of CD4+ T cells and NK cells, effectively increasing the number and activity of CD4+ T cells and NK cells in the tumor microenvironment. At the same time, it can also inhibit the proliferation of Treg cells, reduce the number and activity of Treg cells, and thus increase the synergistic effect of interleukin-2, interleukin-12 and various chemokines. It can significantly improve the tumor immune microenvironment in patients with malignant tumors, thereby enhancing the sensitivity to immunotherapy and improving the efficacy of immunotherapy. It can be used to prepare adjuvant drugs for tumor immunotherapy.
[0018] 2. This invention demonstrates through biological experiments that small molecule peptides have an immunosensitizing effect, enhancing the function of the immune system at the animal level. When used in combination with drugs such as PD1 inhibitors, they can effectively reduce the tumor volume of melanoma, esophageal squamous cell carcinoma, and colonic adenocarcinoma, showing significant therapeutic effects. Furthermore, these small molecule peptides have few toxic side effects, providing a safe and reliable new option for clinical use. Attached Figure Description
[0019] Figure 1 These are in vitro photographs and volume comparisons of melanomas in mice from different treatment groups.
[0020] Figure 2 This is a flow cytometry analysis result of T cells in melanoma tissues of mice from different treatment groups;
[0021] In the figure, A represents the expression of CD4+ T cells, and B represents the expression of Treg cells.
[0022] Figure 3 This is a flow cytometry analysis result of NK cells in melanoma tissues of mice from different treatment groups.
[0023] Figure 4 These are in vitro photographs and volume comparisons of colon tumors in mice from different treatment groups.
[0024] Figure 5 These are in vitro photographs and volume comparisons of esophageal tumors in mice from different treatment groups.
[0025] Figure 6 This is a flow cytometry analysis result of T cells in esophageal tumor tissues of mice in different treatment groups.
[0026] Figure 7 This is a comparison of RNA-seq results of relevant mononuclear cells in the subcutaneous xenograft tumors of mice in Example 2;
[0027] In the figure, A is a KEGG enrichment map of mononuclear cells of mice in different groups, and B is a heatmap of some immune-related pathways. Detailed Implementation
[0028] The technical solution of the present invention will be further described below with reference to embodiments, but the scope of protection of the present invention is not limited thereto. Unless otherwise specified, the reagents and materials involved in the embodiments are all commercially available products.
[0029] The embodiments of this invention have been approved by the Medical Ethics Committee of the Second Hospital of Shandong University.
[0030] The PD1 inhibitor described in this example is available from Leinco.
[0031] The melanoma cells used in the examples are available from Shangen Biotechnology Co., Ltd., the MC38 colon adenocarcinoma cells are available from Fuheng Biotechnology Co., Ltd., and the mEC25 esophageal cancer cells are available from Yaji Biotechnology Co., Ltd.
[0032] Example 1: Preparation and Properties of Small Molecule Peptides
[0033] A small molecule polypeptide, T-P28, was synthesized by Zhejiang Paipai Biotechnology Co., Ltd. using a solid-phase synthesis method according to the amino acid sequence shown in SEQ ID NO.1. The specific sequence is as follows:
[0034] N-Acetyl-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-Lys-Asp-Leu-Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn-OH.
[0035] Alphabetical order:
[0036] N-Acetyl-SDAAVDTSSEITTKDLKEKKE-VVEEAEN-OH.
[0037] Properties and solubility: The small molecule polypeptides obtained in this example are white, in the form of powder or loose lumps, soluble in trifluoroacetic acid, and slightly soluble in water.
[0038] Clarity and color of the solution: Add the small molecule polypeptide obtained in this example to a 0.02 mol / L phosphate buffer solution (pH=7.0) and dissolve it to prepare a solution containing 1.6 mg per 1 mL. The solution should be clear and colorless.
[0039] Pharmacokinetic properties: In healthy individuals, a single subcutaneous injection of 1.6 mg of the immunosensitizing small molecule polypeptide resulted in a peak plasma concentration of approximately 37.51 ng / mL, a time to peak concentration of approximately 1.67 hours, an AUC0-15 of approximately 152.15 ng / mL·h, and a half-life of approximately 1.65 hours.
[0040] Toxic side effects: The genotoxicity test results of the small molecule polypeptide obtained in this embodiment were negative.
[0041] Reproductive toxicity: The small molecule polypeptides obtained in this example have no significant teratogenic effect on animal fetuses.
[0042] Storage: Protect from light, seal, and store at 2–8℃.
[0043] The small molecule peptide was dissolved in sterile water for injection to obtain a small molecule peptide solution with a concentration of 0.05 mg / ml, which was used as the small molecule peptide injection in the subsequent examples.
[0044] Example 2: The role of small molecule peptides in melanoma immunotherapy
[0045] 1. Twenty-eight C57BL / 6J mice were randomly divided into four groups: a control group, an anti-PD1 group (PD1), an immunosensitizing peptide group (T-P28), and an immunosensitizing peptide + anti-PD1 group (T-P28 + PD1). The immunosensitizing peptide group and the immunosensitizing peptide + anti-PD1 group were administered a subcutaneous injection of the small molecule peptide described in Example 1 before tumor formation, at a dose of 0.25 mg / kg once daily.
[0046] Then, a subcutaneous melanoma (B16-F10) xenograft model was constructed in mice belonging to the control group, anti-PD1 group, immunosensitizing peptide group, and immunosensitizing peptide + anti-PD1 group. Specifically, mice were subcutaneously injected with B16-F10 melanoma cells at a dose of 3 × 10⁻⁶. 5Each mouse was injected with a PD1 inhibitor subcutaneously after tumor formation. The PD1 inhibitor was administered subcutaneously to the anti-PD1 group at a dose of 150 μg / mouse every 3 days. The small molecule polypeptide injection described in Example 1 was administered subcutaneously to the immunosensitizing polypeptide group at a dose of 0.25 mg / kg every 3 days. The PD1 inhibitor and the small molecule polypeptide injection described in Example 1 were administered subcutaneously to the immunosensitizing polypeptide + anti-PD1 group at a dose of 150 μg / mouse every 3 days.
[0047] 2. After 19 days of feeding, the four groups of mice from step 1 were dissected, melanomas were removed, and the size of the melanomas was observed and measured. The results are as follows: Figure 1 As shown.
[0048] Depend on Figure 1 The results showed that, compared to the control group, the melanoma volume was significantly reduced in the anti-PD1 group (PD1), the immunosensitizing peptide group (T-P28), and the immunosensitizing peptide + anti-PD1 group (T-P28 + PD1). Furthermore, compared to the anti-PD1 group (PD1), the melanoma volume in the immunosensitizing peptide + anti-PD1 group (T-P28 + PD1) was even smaller. This indicates that the small molecule peptide T-P28 has immunosensitizing function, and its combined use with a PD1 inhibitor results in a more significant therapeutic effect on melanoma, demonstrating reduced heterogeneity in immunotherapy.
[0049] 3. After thoroughly grinding the melanoma tissue, flow cytometry was used to statistically analyze the relative numbers of T cell subsets and NK cells. The results are as follows: Figures 2-3 As shown.
[0050] Depend on Figures 2-3 It was found that, compared with the anti-PD1 group (PD1), the immunosensitizing peptide + anti-PD1 group (T-P28 + PD1) showed a significant increase in the number of CD4+ T cells and a significant decrease in the number of Treg cells, while the number of NK cells and their cytokines also increased significantly. This indicates that the small molecule peptide T-P28 can promote the proliferation of CD4+ T cells and NK cells, inhibit the proliferation of Treg cells, improve the tumor immune microenvironment of melanoma, thereby enhancing the sensitivity of melanoma immunotherapy and improving the efficacy of melanoma immunotherapy. It can be used to prepare adjuvant drugs for melanoma immunotherapy.
[0051] Example 3: The role of small molecule peptides in immunotherapy for colon adenocarcinoma
[0052] 1. Twenty C57BL / 6J mice were randomly divided into four groups: a control group, an anti-PD1 group (PD1), an immunosensitizing peptide group (T-P28), and an immunosensitizing peptide + anti-PD1 group (T-P28 + PD1). The immunosensitizing peptide group and the immunosensitizing peptide + anti-PD1 group were administered a subcutaneous injection of the small molecule peptide described in Example 1 before tumor formation, at a dose of 0.25 mg / kg once daily.
[0053] Then, a subcutaneous xenograft model of colon adenocarcinoma (MC38) was constructed in mice belonging to the control group, anti-PD1 group, immunosensitizing peptide group, and immunosensitizing peptide + anti-PD1 group. Specifically, mice were subcutaneously injected with MC38 colon adenocarcinoma cells at a dose of 5 × 10⁻⁶. 5 Each mouse was injected with a PD1 inhibitor subcutaneously after tumor formation. The PD1 inhibitor was administered subcutaneously to the anti-PD1 group at a dose of 150 μg / mouse every 3 days. The small molecule polypeptide injection described in Example 1 was administered subcutaneously to the immunosensitizing polypeptide group at a dose of 0.25 mg / kg every 3 days. The PD1 inhibitor and the small molecule polypeptide injection described in Example 1 were administered subcutaneously to the immunosensitizing polypeptide + anti-PD1 group at a dose of 150 μg / mouse every 3 days.
[0054] 2. After 15 days of feeding, the four groups of mice from step 1 were dissected, colon tumors were removed, and the size of the colon tumors was observed and measured. The results are as follows: Figure 4 As shown.
[0055] Depend on Figure 4 The results showed that, compared to the control group, the volume of colon tumors in the anti-PD1 group (PD1), the immunosensitizing peptide group (T-P28), and the immunosensitizing peptide + anti-PD1 group (T-P28 + PD1) were significantly smaller. Furthermore, compared to the anti-PD1 group (PD1), the volume of colon tumors in the immunosensitizing peptide + anti-PD1 group (T-P28 + PD1) was even smaller. This indicates that the small molecule peptide T-P28 has immunosensitizing function, and its combined use with a PD1 inhibitor results in a more significant therapeutic effect on colon tumors, demonstrating reduced heterogeneity in immunotherapy.
[0056] Example 4: The role of small molecule peptides in esophageal cancer immunotherapy
[0057] 1. Sixteen C57BL / 6J mice were randomly divided into four groups: a control group, an anti-PD1 group (PD1), an immunosensitizing peptide group (T-P28), and an immunosensitizing peptide + anti-PD1 group (T-P28 + PD1). The immunosensitizing peptide group and the immunosensitizing peptide + anti-PD1 group were administered a subcutaneous injection of the small molecule peptide described in Example 1 before tumor formation, at a dose of 0.25 mg / kg once daily.
[0058] Then, a subcutaneous xenograft model of esophageal cancer (mEC25) was constructed in mice belonging to the control group, anti-PD1 group, immunosensitizing peptide group, and immunosensitizing peptide + anti-PD1 group. Specifically, mice were subcutaneously injected with mEC25 esophageal cancer cells at a dose of 3 × 10⁻⁶. 6 Each mouse was injected with a PD1 inhibitor subcutaneously after tumor formation. The PD1 inhibitor was administered subcutaneously to the anti-PD1 group at a dose of 150 μg / mouse every 3 days. The small molecule polypeptide injection described in Example 1 was administered subcutaneously to the immunosensitizing polypeptide group at a dose of 0.25 mg / kg every 1 day. The PD1 inhibitor and the small molecule polypeptide injection described in Example 1 were administered subcutaneously to the immunosensitizing polypeptide + anti-PD1 group at a dose of 150 μg / mouse every 3 days.
[0059] 2. After 48 days of rearing, the four groups of mice from step 1 were dissected, esophageal tumors were removed, and the size of the esophageal tumors was observed and measured. The results are as follows: Figure 5 As shown.
[0060] Depend on Figure 5 The results showed that, compared to the control group, the esophageal tumor volume was significantly reduced in the anti-PD1 group (PD1), the immunosensitizing peptide group (T-P28), and the immunosensitizing peptide + anti-PD1 group (T-P28 + PD1). Furthermore, compared to the anti-PD1 group (PD1), the esophageal tumor volume in the immunosensitizing peptide + anti-PD1 group (T-P28 + PD1) was even smaller. This indicates that the small molecule peptide T-P28 has immunosensitizing function, and its combined use with a PD1 inhibitor results in a more significant therapeutic effect on esophageal tumors, demonstrating reduced heterogeneity in immunotherapy.
[0061] 3. After thoroughly grinding the esophageal tumor tissue, flow cytometry was used to statistically analyze the relative numbers of T cell subsets. The results are as follows: Figure 6 As shown.
[0062] Depend on Figure 6It was found that, compared with the anti-PD1 group (PD1 group), the number of Treg cells in the immunosensitizing peptide + anti-PD1 group (T-P28 + PD1) was significantly reduced. This indicates that the small molecule peptide T-P28 can inhibit Treg cell proliferation, improve the tumor immune microenvironment of esophageal tumors, thereby enhancing the sensitivity of esophageal tumor immunotherapy and improving the efficacy of esophageal tumor immunotherapy. It can be used to prepare adjuvant drugs for esophageal tumor immunotherapy.
[0063] Example 5: Changes in immune cells after combined application of immunosensitizing small molecule peptides and PD1 inhibitors using RNA-seq analysis. Mononuclear cells were collected from subcutaneous xenograft tumors of four groups of mice after 19 days of feeding in step 2 of Example 2. The mononuclear cells of the anti-PD1 group (PD1) were divided into two subgroups, resulting in a total of five groups of mononuclear cells: immunosensitizing peptide group (T-P28), anti-PD1 poor efficacy group (PD1-PR), anti-PD1 good efficacy group (PD1-PD), and immunosensitizing peptide + anti-PD1 group (T-P28+PD1). KEGG enrichment analysis was then performed on these five groups of mononuclear cells using RNA-seq. The results are as follows: Figure 7 As shown in Figure A, a heatmap was created to display the enrichment pathways related to immune cells, and the results are as follows. Figure 7 As shown in B.
[0064] Depend on Figure 7 It can be seen that the results of the immunosensitizing peptide + anti-PD1 group are similar to those of the anti-PD1 effective group. Compared with the control group and the anti-PD1 poor effective group, the expression of T cell activation, NK cell, B cell, chemokines and related genes such as interleukin-2 and interleukin-12 is significantly increased.
[0065] The embodiments described above are merely preferred implementations of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.
Claims
1. A small molecule polypeptide, characterized in that, The amino acid sequence is shown in SEQ ID NO.
1.
2. The use of the small molecule polypeptide of claim 1 in promoting the proliferation of CD4+ T cells and NK cells and inhibiting the proliferation of Treg cells in vivo and / or in vitro.
3. The use of the small molecule polypeptide of claim 1 in the preparation of a medicament for the prevention and / or treatment of diseases by regulating CD4+ T cells, NK cells and Treg cells.
4. The use of the small molecule polypeptide according to claim 1 in the preparation of adjuvant drugs for tumor immunotherapy.
5. The application as described in claim 4, characterized in that, The tumor is melanoma, colon cancer, or esophageal cancer.
6. The application as described in claim 4, characterized in that, The adjuvant drug for tumor immunotherapy is used in combination with drugs or therapies for the prevention and / or treatment of tumor immune tolerance.
7. The application as described in claim 4, characterized in that, The adjuvant drugs for tumor immunotherapy also include pharmaceutically acceptable carriers, excipients, additives, supplements, dispersants, or stabilizers.
8. The application as described in claim 4, characterized in that, The adjuvant drugs for tumor immunotherapy are solutions, emulsions, suspensions, or injections.